Cytokine binding proteins (soluble cytokine receptors) are usually the extracellular ligand binding domains of their respective cell surface cytokine receptors. They are produced either by alternative splicing or by proteolytic cleavage of the cell surface receptor. These soluble receptors have been described in the past: for example, the soluble receptors for IL-6 and IFNγ (Novick et al. 1989), TNF (Engelmann et al. 1989 and Engelmann et al. 1990), IL-1 and IL-4 (Maliszewski et al. 1990) and IFNα/β (Novick et al. 1994, Novick et al. 1992). One cytokine-binding protein, named osteoprotegerin (OPG, also known as osteoclast inhibitory factor—OCIF), a member of the TNFR/Fas family, appears to be the first example of a soluble receptor that exists only as a secreted protein (Anderson et al. 1997, Simonet et al. 997, Yasuda et al. 1998).
An interleukin-18 binding protein (IL-18BP) was affinity purified, on an IL-18 column, from urine (Novick et al. 1999). IL-18BP abolishes IL-18 induction of IFNγy, and IL-18 activation of NF-kB in vitro. In addition, IL-18-BP inhibits induction of IFNγ in mice injected with LPS. The IL-18BP gene was localized to the human chromosome 11, and no exon coding for a transmembrane domain could be found in the 8.3 kb genomic sequence comprising the IL-18BP gene. Four isoforms of IL-18BP generated by alternative mRNA splicing have been found in humans so far. They were designated IL-18BP a, b, c, and d, all sharing the same N-terminus and differing in the C-terminus (Novick et al 1999). These isoforms vary in their ability to bind IL-18 (Kim et al. 2000). Of the four, human IL-18BP (hIL-18BP) isoforms a and c are known to have a neutralizing capacity for IL-18. The most abundant IL-18BP isoform, the spliced variant isoform a, exhibits a high affinity for IL-18 with a rapid on-rate and a slow off-rate, and a dissociation constant (Kd) of approximately 0.4 nM (Kim et al. 2000). IL-18BP is constitutively expressed in the spleen (Novick 1999), and circulates at plasma concentrations of 2.5 ng/ml (Novick et al. 2001). The residues involved in the interaction of IL-18 with IL-18BP have been described through the use of computer modelling (Kim et al. 2000) and based on the interaction between the similar protein IL-1β with the IL-1R type I (Vigers et al. 1997). According to the model of IL-18 binding to the IL18BP, the Glu residue at position 42 and Lys residue at position 89 of IL-18 have been proposed to bind to Lys-130 and Glu-114 in IL-18BP, respectively (Kim et al. 2000).
As mentioned, IL-18 induces IFNγ which, in turn, was recently reported to induce IL-18BPa mRNA generation in vitro (Muhl et al 2000). Therefore, IL-18BPa could serve as a “shut off” signal, terminating the inflammatory response.
IL-18BP is significantly homologous to a family of proteins encoded by several Poxviruses (Novick et al. 1999, Xiang and Moss 1999). Inhibition of IL-18 by this putative viral IL-18BP may attenuate the inflammatory antiviral Th1 response. Serum IL 18BP is significantly elevated in sepsis, indicating its role in regulating immune responses in vivo (Novick et al. 2001). Indeed, IL-18BP is induced by IFNγ in various cells, suggesting that it serves as a negative feedback inhibitor of the IL-18-mediated immune response (Mughl et al. 2000)
Preliminary results indicate that IL-18BP mRNA is detected in leukocytes, colon, small intestine, prostate and particularly in spleen cells (Novick et al. 1999). The component cells of the spleen consist of macrophages, lymphocytes, and plasma cells with additional cells derived from the circulation.
The activity of elements that control transcription, promoter and enhancers vary considerably among different cell types. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (reviewed in Dynan and Tjian 1985, McKnight and Tjian 1986, Sassone-Corsi and Borreli 1986 and Maniatis et al 1987). The combination of different recognition sequences and the amounts of the cognate transcription factors determine efficiency with which a given gene is transcribed in a particular cell type. Many eukaryotic promoters contain two types of recognition sequences: the TATA box and the upstream promoter elements. The TATA box, located 25-30 bp upstreaqm of the transcription initiation site, is thought to be involved in directing RNA polymerase II to begin RNA synthesis at the correct site. In contrast, the upstream promoter elements determine the rate at which transcription is initiated. Enhancer elements can stimulate transcription up to 1000-fold from linked homologous or heterologous promoters. However unlike upstream promoter elements, enhancers are active when placed downstream from the transcription initiation site or at considerable distance from the promoter. Many enhancers of cellular genes work exclusively in a particular tissue or cell type (reviewed by Voss et al. 1986, Maniatis et al. 1987). In addition some enhancers become active only under specific conditions that are generated by the presence of an inducer, such as a hormone or metal ion (reviewed by Sassone-Corsi and Borrelli 1986 and Maniatis 1987). Because of these differences in cell specificities of cellular enhancers, the choice of promoter and enhancer elements to be incorporated into a eukaryotic expression vector will be determined by the cell types in which the recombinant gene is to be expressed. Conversely, the use of a prefabricated vector containing a specific promoter and cellular enhancer may severely limit the cell types in which expression can be obtained.
Many enhancer elements derived from viruses have a broader host range and are active in a variety of tissues, although significant quantitative differences are observed among the different cell typees. For example, the SV40 early enhancer is promiscuously active in many cell types derived from a variety of mammalian species, and vectors incorporating this enhancer have consequently been used (Dijkema et al. 1985). Two other enhancer/promoter combinations that are active in a broad range of cells are derived from the long repeat (LTR) of the Rous sarcoma virus genome (Gorman et al 1982b) and from human cytomegalovirus (Boshart et al. 1985).